Protein glycosylation, the attachment of carbohydrates to proteins, is one of the most common modifications found in eukaryotics. Glycosylation falls into three categories: N-linked modification of the asparagine (Asn) side chain, O-linked modification of serine (Ser) or threonine (Thr) and the modification of the protein C-carboxyl terminus by glycosylphosphatidyl inositol (GPI) derivatization. O-linked glycosylation and GPI anchor derivatization are post-translational modifications that take place in the Golgi. On the other hand, N-linked glycosylation is a co-translational modification. As proteins are synthesized, the polypeptide enters the endoplasmic reticulum, where oligosaccharyl transferase (OT) attaches a branched carbohydrate (N-glycan) to the side chain of certain asparagine residues (Hirschberg, C. B., Snider, M. D. 1987. Annu Rev Biochem. 56, 63-87.) This process requires an Asn-X-Ser/Thr consensus sequence in the peptide substrate, where X is any amino acid except proline (Bause, E. 1983. Biochem J. 209, 331-6; Marshall, R. D. 1972. Annu Rev Biochem. 41, 673-702.) The attached glycans, are subsequently modified by a complex array of glycosidases and glycosyl transferases in the endoplasmic reticulum (ER) and Golgi apparatus. The attached glycans play an important role in protein folding, as well as directing the protein to the appropriate location within the cell (Dwek, R. A. 1996. Chem. Rev. 96, 683-720; O'Connor, S. E., Imperiali, B. 1996. Chem. Biol. 3, 803-12.) Outside the cell, the sugars aid in protein-protein interactions, often modulating the activity of the protein to which they are attached. Depending on the glycan composition, they can also protect against or facilitate protein degradation in circulation, as well as target the protein to a specific organ (Crocker, P. R., Varki, A. 2001. Immunology. 103, 137-45; Helenius, A., Aebi, M. 2001. Science. 291; 2364-9; Imperiali, B., O'Connor, S. E. 1999. Curr Opin Chem. Biol. 3, 643-9.)
Glycans also have an important role in normal biology, as evidenced by the high lethality in cases of defective glycosylation. In mouse knockout models, disrupting even one of the biosynthetic enzymes can lead to enormous multisystemic disorders, and several result in embryonic lethality (Furukawa, K., et al. 2001. Biochim Biophys Acta. 1525, 1-12.) There are currently six recognized human congenital disorders of glycosylation (CDGs), all resulting in patients with multiple organ abnormalities, developmental delay and immune problems, among others (Jaeken, J., Matthijs, G. 2001. Annu Rev Genomics Hum Genet. 2, 129-51; Freeze, H. H., Aebi, M. 1999. Biochim Biophys Acta. 1455, 167-78; Carchon, H., et al. 1999. Biochim Biophys Acta. 1455, 155-65.) In fact, the immune system is one of the most commonly studied systems where glycans, such as N-glycans, have been shown to play an important physiological role. For example, specific carbohydrate structures are recognized by selectins, a family of proteins expressed on endothelial cells or lymphocytes that can trigger the immune system upon activation (Powell, L. D., et al. J Biol. Chem. 268, 7019-27; Sgroi, D., et al. 1993. J Biol. Chem. 268, 7011-8.) The same class of structures that are necessary for proper immune function can also provide a binding site for certain viruses, bacteria or tumor cells in the body (Karlsson, K. A. 1998. Mol. Microbiol. 29, 1-11; Pritchett, T. J., et al. 1987. Virology. 160, 502-6.)
Viral infection is mediated by the interaction of viral proteins with glycans on the cell surfaces of the host (Van Eijk, M., et al. 2003. Am J Respir Cell Mol. Biol. 6, 871-9.) Despite the increasing evidence associating glycans to different pathogenic conditions, in multiple instances it is unclear whether changes in glycan structure are a cause or a symptom of the disorder. In cystic fibrosis, increased antennary fucosylation (α1-3 linked to GlcNAc) is observed on surface membrane glycoproteins of airway epithelial cells (Glick, M. C., et al. 2001. Biochimie. 83, 743-7; Scanlin, T. F., Glick, M. C. 2000. Glycoconj. J 17, 617-26.)
There have also been many reports of alterations in glycan composition on cancer cell proteins. For example, there are indications that prostate cancer cells produce prostate specific antigen (PSA) with more glycan branching than non-cancer cells (Peracaula, R., et al. 2003. Glycobiology. 13, 457-70; Belanger, A., et al. 1995. Prostate. 27, 187-97; Prakash, S., Robbins, P. W. 2000. Glycobiology. 10, 173-6.) Melanoma and bladder cancer cells produce proteins with highly branched glycans due to an overexpression of the biosynthetic enzyme β1,6-N-acetyl-glucosaminyltransferase V (GnT-V) (Chakraborty, A. K., et al. 2001. Cell Growth Differ. 12, 623-30; Przybylo, M., et al. 2002. Cancer Cell Int. 2, 6.) Increased sialylation and additional branching have also been observed in cells from human breast and colon neoplasia (Lin, S., et al. 2002. Exp Cell Res. 276, 101-10; Nemoto-Sasaki, Y., et al. 2001. Glycoconj J. 18, 895-906; Dennis, J. W., et al. 1999. Biochim Biophys Acta. 1473, 21-34; Fernandes, B., et al. 1991. Cancer Res. 51, 718-23.)